Porous gelatin material, gelatin structures, methods for preparation of the same and uses thereof

ABSTRACT

The present invention relates to a porous gelatin material in the form of spherical particles with a continuous pore structure and cast, three-dimensional, porous gelatin structures. The invention also comprises methods for preparation of the porous gelatin materials and structures. The method for preparing the porous gelatin material in the form of spheres with a continuous pore structure comprises the steps of preparing a homogenous water-based gelatin solution, adding an emulsifier with an HLD value &gt;9, adding a first composition comprising an organic solvent and an emulsifier with an HLB value &gt;9, adding a second composition comprising an organic solvent and an emulsifier with an HLB value &lt;8 and allowing the gelatin material to solidify. Uses of the materials according to the invention are also included.

FIELD OF THE INVENTION

The present invention relates to a porous gelatin material with acontinuous structure in the form of spherical particles, cast,three-dimensional, porous gelatin structures, methods for producing thesame and uses thereof.

BACKGROUND ART

Most animal cells are surface-dependent, that is they have to beattached to a surface to be able to survive and/or proliferate.Traditionally this surface has been the interior of glass or plasticflasks. Great difficulties have been involved in culture of cells on alarge scale or in implantation of the cells. The size of these cells is5-20 μm.

Microcarriers are small particles of 0.2 mm diameter, to which the cellscan attach and on which they can proliferate (van Wezel, A. L. Nature216 (1967) 64-65 Growth of cell strains and primary cells onmicrocarriers in homogeneous culture). These particles have to someextent made it easier to culture surface-dependent cells on a largescale.

The most common type of microcarrier consists of spherical carriers madeof dextran and modified by derivatisation with positive groups. Thismakes the cells adhere to the carriers. Another way of making the cellsadhere is either to produce the carriers of gelatin or to link gelatinto the surface of dextran particles. Gelatin is made of collagen whichis the substance to which cells normally adhere. The carriers that arecurrently available for cells are not optimal in every respect. Thesecarriers are often homogeneous, that is the cells can only adhere/growon their surface. As a result, the surface available for celladhesion/cell growth will be limited to the surface area of thecarriers. Furthermore, the cells can only adhere/grow in two dimensionsin comparison to normally three dimensions in vitro. Another limitationof prior-art systems is that when the carriers are used for culture inculture vessels the cells will be damaged by the forces caused by thestirring system.

To some extent this has already been solved by preparing particleshaving a great number of encased cavities by means of an emulsion method(Kjell Nilsson and Klaus Mosbach, Swedish Patent 8504764-5, Macroporousparticles, method for its production and use of the same). This patentspecification discloses how particles having a great number of encasedcavities can be prepared by adding to an aqueous solution of the matrixmaterial a solid, liquid or gaseous cavity-forming compound. After theparticles have formed by dispersion in a water-insoluble dispersingagent, the matrix is made water-insoluble by cooling, covalentcrosslinking or polymerisation. The cavity-forming compound is removedto obtain the encased cavities.

The particles can be used as ion exchangers, gel filter media,chromatography media and microcarriers in cell culture. The matrix ismade of protein, polysaccharide or polyacrylamide.

The invention according to Swedish Patent 8504764-5 provided particlesin which some of the cavities of the particles were available for celladhesion/cell growth. It has however been found that the thus-obtainedparticles were not optimal in some respects. Optimally all the cavitiesare interconnected so that a continuous porous phase and a continuousmatrix phase are obtained. In the present invention, this state wasunexpectedly obtained in the case of gelatin by a combination ofemulsifier and solvent.

In addition, this state has made it possible to prepare both particlesand other three-dimensional shapes.

This phase separation must be stable on a micro-level for the length oftime necessary for the preparation of the desirable shapes. Furthermorethe phase separation must not result in a separation of the phases on amacro-level since this yields shapes without porosity.

SUMMARY OF THE INVENTION

According to one aspect, the present invention relates to a method forpreparation of a porous gelatin material, in the form of spheres, with acontinuous pore structure, the method comprising the steps of: preparinga homogeneous water-based gelatin solution; adding an emulsifier with anHLB value >9; adding a first composition comprising an organic solventand an emulsifier with an HLB value >9; adding a second compositioncomprising an organic solvent and an emulsifier with an HLB value <8;and allowing the gelatin material to solidify.

According to a second aspect, the invention relates to a porous gelatinmaterial, in the form of spherical particles, with a continuous porestructure produced by preparing a homogeneous water-based gelatinsolution; adding an emulsifier with an HLB value >9; adding a firstcomposition comprising an organic solvent and an emulsifier with an HLBvalue >9; adding a second composition comprising an organic solvent andan emulsifier with an HLB value <8; and allowing the gelatin material tosolidify.

According to a further aspect, the invention relates to a method forproducing a cast, three-dimensional, porous gelatin structure which canbe obtained by preparing a homogenous water-based gelatin solution;adding an emulsifier with an HLB value >9; adding a first compositioncomprising an organic solvent and an emulsifier with an HLB value >9;and casting the gelatin solution in a mould.

According to another aspect, the invention relates to a cast,three-dimensional, porous gelatin structure which can be obtained bypreparing a homogenous water-based gelatin solution; adding anemulsifier with an HLD value >9; adding a first composition comprisingan organic solvent and an emulsifier with an HLD value >9; and castingthe gelatin solution in a mould.

According to yet another aspect, the invention relates to use of aporous gelatin material or a cast, three-dimensional, porous gelatinstructure produced according to the present invention, as carrier forcells.

According to a further aspect, the invention relates to use of a porousgelatin material or a cast, three-dimensional, porous gelatin structureproduced as described above for making an implant.

Another aspect of the present invention involves a method for implantinga biocompatible, porous gelatin material as described above or a cast,three-dimensional, porous gelatin structure as described above ascarrier for cells in an individual for production of substances,comprising implanting in said biocompatible, porous gelatin material orsaid cast, three-dimensional, porous gelatin structure in the individualand subsequently allowing the cells on the biocompatible, porousmaterial or the cast, three-dimensional, porous gelatin structure toproduce said substances.

DETAILED DESCRIPTION OF THE INVENTION

According to one embodiment of the invention, the above methods furthercomprise the step of chemically crosslinking the gelatin material. Thiscrosslinking can be carried out with poly- or bifunctional isocyanatecompounds, such as hexamethylenediisocyanate or toluene-diisocyanate,poly- or bifunctional aldehydes, such as glutardialdehyde. Gelatin canalso be crosslinked with formaldehyde in liquid form or in gaseous form.

According to another embodiment, the emulsifier is selected with an HLBvalue >9 from, but not exclusively, the group consisting of Tween 80(polyoxysorbitan mono-oleate, HLB value=15), Tween 40 (polyoxyethylenestearic acid, HLB value=15), Myrj 52 (polyoxyethylene stearic acid, HLBvalue=17) and Brij 58 (polyoxyethylene cetyl alcohol HLB value=16).

According to yet another embodiment, the emulsifier is selected with anHLB value <8 from, but not exclusively, the group consisting of Span 85(HLB value=2), Span 65 (HLB value=2) and Atmos 300 (HLB value=2.5).

“Atmos” is the trademark of a series of mono- and diglycerideemulsifiers which are used in ice cream and frozen desserts (TheCondensed Chemical Dictionary, 6th Edition, 1961, p. 116).

According to another embodiment, the organic solvent is selected from,but not exclusively, the group consisting of cyclohexane, toluene,paraffin oil and industrial oil.

According to a particularly preferred embodiment according to theinvention, the organic solvent is cyclohexane.

The selection of emulsifier in the present methods according to theinvention is not critical as long as the requirement on the HLB value ofthe emulsifier is fulfilled. It will be obvious to the one skilled inthe art which emulsifiers can be selected as arbitrary agents.

Emulsifiers are characterized by the hydrophilic-lipophilic balancebeing stated in the form of HLB values. They usually vary between 1 and20. A low HLB value indicates that the lipophilic part of the emulsifierdominates and a high value that the emulsifier has primarily hydrophilicproperties (Galenisk farmaci, Erik Sandell, 3^(rd) Edition, 1982, p.97).

In 8504764-5, the pore structure is produced by emulsifying, in one ofthe steps of the preparation, an organic solvent in the homogeneousgelatin solution. This emulsion is stabilized with an emulsifier, forinstance Span 85, with an HLB value <8. This type of emulsifier yieldsstable emulsions of solvent in water phases. After cooling below thesolidification point of the gelatin solution, the solvent is removed.The result is a material having a great number of encased cavities whichare not interconnected.

According to the present invention, a continuous pore structure isprepared by adding to a homogeneous water-based gelatin solution, in oneof the steps of the preparation process, a solvent, for instancecyclohexane, containing an emulsifier with an HLB value >9. This type ofemulsifier yields stable emulsions of water phases in solvents. However,since the volume of the water phase is much greater than the volume ofthe solvent phase, this results in an unstable proportion. This unstableproportion results in an unexpected microscopic phase separation whichis stable for a length of time which is sufficient to prepare thedesirable shapes. The addition of an emulsifier with an HLB value >9 tothe gelatin phase facilitates the microscopic phase separation. Anotherfeature distinguishing the present invention from prior-art technique isthat the present invention only works when gelatin is used asmatrix-forming compound. Experiments carried out with polysaccharidesand polyacrylamide do not result in any microscopic phase separation.Thus using present-day knowledge, the new invention only works withgelatin as matrix-forming compound. Therefore something unique andunexpected happens in the combination of gelatin and the emulsifiersystem. As mentioned above, the invention disclosed in 8504764-5provides encased cavities in a great number of matrix-forming materialsand since the cavities of the particles are not interconnected, themaximum number of cells cannot adhere/grow. In 8504764-5 discretedroplets/pores are thus obtained, but according to the present inventiontwo continuous phases are obtained, one gelatin-water phase and onesolvent phase, which in the solidification provide a continuous porestructure.

The addition of the first composition to the gelatin solution results ina phase separation at a given temperature. The different phases are notvisible with the naked eye but the mixture becomes whitish. According toone embodiment of the present invention, three-dimensional gelatinstructures can be obtained by casting the gelatin solution, after addingthe first composition to the gelatin solution, in a mould. The mould canbe any type of mould and adjusted to the intended final use of thegelatin structure. For instance, the gelatin structure is cast to tubes,ears or other in-vivo-like structures, such as fingers, toes, nipples ornoses.

The addition of the second composition (cyclohexane containing Span 85)to the gelatin solution yields droplets surrounded by solvent, saiddroplets being stabilised by Span 85. These droplets contain a gelatinphase and a solvent phase. Both phases are continuous. The addition ofthe second composition results in spherical particles.

According to another embodiment of the invention, the biocompatible,porous material or the cast, three-dimensional gelatin structure is usedto culture artificial skin, artificial organs, fatty tissue and bloodvessels.

The biocompatible, porous material according to the present inventioncan be used both as carrier for cells in cell culture and as carrier forexisting cells for the production of a desirable substance before/afterimplantation in an individual. The cells can be either the individual'sown cells or cells from another source (characteristic of the species orforeign to the species). In some cases the cells as such can be thedesirable product, for instance attached initial stages of adipocytes(preadipocytes) on the carrier which after implantation can proliferateso as then to be converted into adipocytes (fat cells). One field ofapplication is for instance plastic surgery.

It is also possible to implant the produced porous structures accordingto the invention in a human body without the addition of cells. Afterimplantation, the neighbouring cells in the body will migrate into andcolonise the structure. After the implanted structure has dissolved, thecolonised cells will have formed a structure corresponding to theimplant. The cross-linking degree of the gelatin material controls thetime it takes for the gelatin structure to dissolve in the body. It isthus possible to control the dissolution for the intended application.An example of this is in plastic surgery where carriers according to theinvention without accompanying cells are injected at the seat of awrinkle. The cells surrounding the carriers migrate towards the carriersand colonise them. Gradually, as the carriers are being dissolved bysurrounding enzymes the migrated cells occupy the seat of the wrinkle.This results in the wrinkle being smoothed out.

Yet another example is to use the material with adhering cells whentesting drugs. By measuring variables which reflect the state of theadhering cells, predictions can be made about the effectiveness/toxicityof the potential drug.

Another example is skin cells on the carrier which can be used fortreating different types of injuries to the skin. Another example ismyoblasts (muscle cells) which can be used in treatment of e.g. cardiacinfarction. One more example is hepatocytes (liver cells) which can beused to render toxic substances in liver lesions harmless. Also morecomplex structures such as islets of Langerhans can be attached toand/or in the porous carrier. Islets of Langerhans are composed of aplurality of different cell types and constitute the system thatregulates the blood sugar level. These islets are considerably largerand require a pore size of the carrier of 50-200 μm.

The term “substance” used herein relates to the substances that can beproduced by different cells or micro-organisms, for instanceantibiotics, pharmaceutical substances, e.g. dopamine which is a keysubstance in Parkinson's disease, and different interferons which areactive substances in treatment of cancer.

The term “porous” used herein in combination with both the sphericalparticles and the cast three-dimensional gelatin structures relates tothe fact that the particles and the structures comprise pores in whichcells can grow.

According to the invention, the degradability of the biocompatible,porous gelatin material is determined by the degree of crosslinking ofthe gelatin. An agent can, for instance, be added to enhance or changethe adhesion of cells to said biocompatible, porous material duringcasting of the dispersion, or the agent can be bound chemically to thepolymer or added later. Agents affecting cell adhesion can be eithersimple molecules or proteins. Examples of the former are positively ornegatively charged substances, such as hexamethylene diamine and aminocapronic acid. Also uncharged structures such as fatty acids can beattached to the matrix. Examples of more complex structures are peptidescontaining the amino acid sequence arginine-glycine-aspargine orderivatives thereof. This sequence promotes the adhesion of cells to thecarrier. Examples of proteins are fibronectin and laminin. Alsonon-defined mixtures of proteins (obtained by extraction of tissues),such as ECM (extracellular matrix), can be used.

To prevent rejection, the gel particles can be encapsulated withadhering cells in another material which serves to prevent cells andproteins of the body from recognizing or reacting with the adheringcells. This material can be a polysaccharide or polymer. The materialthus functions as a kind of mechanical barrier against the proteins andcells of the body.

There are several types of gelatin depending on the preparation methodand raw material used in the production. These gelatin types havedifferent properties which can be used to provide the porous materialwith various properties. Furthermore, the gelatin can be modified beforepreparation to obtain further new and desirable properties. Thismodification can be carried out by means of physical methods, such asfractionation, or chemical reactions.

To obtain a shape suitable for the specific application, the prepareddispersion can be formed into different three-dimensional structuresaccording to prior-art methods. Thus spherical particles can be preparedby an emulsion process and membranes by casting on/between plates.Special structures can be cast in specially made moulds, for instanceears. Finishing processes involving mechanical methods can also be usedto obtain the final structure.

The biocompatible, porous material is prepared so that the cells arepresent both inside the continuous pore structure and outside thebiocompatible, porous material. This results in optimal use of thematerial.

In the present description the expression “carrier for cells” isintended to comprise carriers which can be used in culture of differentcells and carriers which can be used for cells to achieve production ofdesirable substances. The expression “carrier for cells” also includesmedical implants for implantation in the human body. A surprising effectin the preparation of the biocompatible, porous gelatin materialaccording to the invention is that the continuous pores obtained in thematerial are uniformly distributed through the cross-section of thematerial. Thus, a more uniform distribution of the cells in thebiocompatible, porous gelatin material is achieved.

EXAMPLES Example 1 Preparation of Spherical Gelatin Particles with LargeContinuous Pores

While stirring 13 g gelatin is dissolved in 100 ml water by heating to40° C. All subsequent steps are carried out while stirring. To thissolution 21 g Tween 80 (polyoxyethylene(20)sorbitanmonooleate) is added.Tween is the trademark of a series of emulsifiers and surfacetants. Theyare polyoxyethylene derivatives of fatty acid partial esters ofhexitolanhydrides (The Condensed Chemical Dictionary, 6^(th) Edition,1961, p. 1182). The mixture is cooled to 35° C. Then 34 ml cyclohexanecontaining 1 g Tween 80 is added. The mixture is further cooled to 32°C., whereupon 34 ml cyclohexane containing 2 g Span 85(sorbitantrioleate) is added. Span is the trademark of a series ofemulsifiers and surfactants. They are fatty acid partial esters ofhexitolanhydrides (or sorbitan) (The Condensed Chemical Dictionary,6^(th) Edition, 1961, p. 1063). The mixture is further cooled until thegelatin solidifies. Emulsifiers and cyclohexane are removed by washingwith acetone. The particles can be dried by allowing the acetone toevaporate.

Example 2 Preparation of Spherical Gelatin Particles with SmallContinuous Pores

While stirring 13 g gelatin is dissolved in 100 ml water by heating to40° C. All the subsequent steps are carried out while stirring. To thissolution 15 g Tween 80 is added. The mixture is cooled to 35° C. Then 12ml cyclohexane containing 1.5 g Tween 80 is added. The mixture isfurther cooled to 32° C., whereupon 58 ml cyclohexane containing 3 gSpan 85 is added. The mixture is further cooled until the gelatinsolidifies. Emulsifiers and cyclohexane are removed by washing withacetone. The particles can be dried by allowing the acetone toevaporate.

Example 3 Crosslinking Method 1

10 g porous material is mixed with 120 ml acetone and 30 ml water inwhich 480 mg sodium acetate trihydrate is dissolved. The material iscrosslinked for two hours by the addition of 0.45 mlhexamethylenediisocyanate and 0.06 ml triethylamine. The crosslinkedparticles are washed with water and acetone. The particles are dried byallowing the acetone to evaporate.

Example 4 Crosslinking Method 2

10 g porous material is mixed with 400 ml water in which 7.2 g sodiumphosphate is dissolved. The material is crosslinked for one hour by theaddition of 0.8 ml hexamethylenediisocyanate and 5 μl triethylamine. Thecrosslinked particles are washed with water and acetone. The particlesare dried by allowing the acetone to evaporate.

In the production of the material above, larger continuous pores can beobtained by using a higher Tween concentration. By increasing the amountof cyclohexane, larger pores are also obtained and simultaneously alarger total pore volume is obtained. By increasing the stirring rate,smaller pores are obtained. It is thus easy to vary the porosity of themembrane within wide limits.

The resistance of the biocompatible, porous material to heat, enzymes,etc. is proportional to the crosslinking degree. An increasedconcentration of crosslinking agent, for instance hexamethylenediisocyanate, results in increased resistance. An increased crosslinkingtime also results in increased resistance. The crosslinking reagentsthat are used in the preparation can be, for instance, bifunctional orpolyfunctional, such as diisocyanates and polyisocyanates, but alsoaldehydes can be bi- and polyfunctional. Other prior-art methods ofcrosslinking gelatin can be used.

The crosslinking can take place in the preparation of the particles byadding the crosslinking reagent to the dispersion before forming. As analternative, the structures formed can be crosslinked on a subsequentoccasion since the porous gelatin structure is maintained as the gelatinsolidifies when cooling.

1. A method for preparation of a porous gelatin material in the form ofspheres with a continuous pore structure, the method comprising thesteps: preparing a homogeneous water-based gelatin solution; adding anemulsifier with an HLB value >9; adding a first composition comprisingan organic solvent and an emulsifier with an HLB value >9; adding asecond composition comprising an organic solvent and an emulsifier withan HLB value <8; and allowing the gelatin material to solidify.
 2. Amethod for preparation of a cast, three-dimensional, porous gelatinstructure comprising the steps: preparing a homogenous water-basedgelatin solution; adding an emulsifier with an HLB value >9; adding afirst composition comprising an organic solvent and an emulsifier withan HLB value >9; and casting the gelatin solution in a mould.
 3. Amethod as claimed in claim 1, further comprising the step of chemicallycrosslinking the gelatin material.
 4. A method as claimed in claim 3,wherein the chemical crosslinking is carried out with poly- orbifunctional isocyanate compounds, such as hexamethylenediisocyanate ortoluenediisocyanate, poly- or bifunctional aldehydes, such asglutardialdehyde, or with formaldehyde.
 5. A method as claimed in claim1, wherein the emulsifier with an HLB value >9 is selected from thegroup consisting of Tween 80, Tween 40, Myrj 52, and Brij
 58. 6. Amethod as claimed in claim 1, wherein the emulsifier with an HLB value<8 is selected from the group consisting of Span 85, Span 65, and Atmos300.
 7. A method as claimed in claim 1, wherein the organic solvent isselected from the group consisting of cyclohexane, toluene, paraffinoils and industrial benzene.
 8. A method as claimed in claim 7, whereinthe organic solvent is cyclohexane.
 9. A porous gelatin material in theform of spheres with a continuous pore structure obtainable by preparinga homogeneous water-based gelatin solution; adding an emulsifier with anHLB value >9; adding a first composition comprising an organic solventand an emulsifier with an HLB value >9; adding a second compositioncomprising an organic solvent and an emulsifier with an HLB value <8;and allowing the gelatin material to solidify.
 10. A cast,three-dimensional, porous gelatin structure obtainable by: preparing ahomogenous water-based gelatin solution; adding an emulsifier with anHLB value >9; adding a composition comprising an organic solvent and anemulsifier with an HLB value >9; and casting the gelatin solution in amould.
 11. A carrier for cells comprising a porous gelatin material or acast three-dimensional porous gelatin structure produced in accordancewith the method of claim
 1. 12. A culture of artificial skin, artificialorgans, or fatty tissue and blood vessels comprising the biocompatibleporous material or a cast three-dimensional porous gelatin structureproduced in accordance with the process of claim
 1. 13. An implantcomprising a porous gelatin material or a cast three-dimensional porousgelatin structure produced in accordance with the method of claim
 1. 14.A method as claimed in claim 10, wherein the cast three-dimensionalgelatin structure is selected among tubes, ears and in-vivo-likestructures.
 15. (canceled)
 16. A method as claimed in claim 2, furthercomprising the step of chemically crosslinking the gelatin material. 17.A method as claimed in claim 11, wherein the cast three-dimensionalgelatin structure is selected among tubes, ears, and in-vivo-likestructures.
 18. A gelatin material according to claim 9, wherein thegelatin has been chemically crosslinked.
 19. A gelatin structureaccording to claim 10, wherein the gelatin has been chemicallycrosslinked.
 20. A method for implanting in an individual a porousgelatin material as claimed in claim 9 as carrier for cells for theproduction of biological substances, comprising introducing such cellsonto said material, implanting said porous gelatin material in theindividual and allowing the cells on said material to produce saidsubstances.
 21. A method for implanting in an individual a porousgelatin material as claimed in claim 18 as carrier for cells for theproduction of biological substances, comprising introducing such cellsonto said material, implanting said porous gelatin material in theindividual and allowing the cells on said material to produce saidsubstances.
 22. A method for implanting in an individual a cast, threedimensional, porous gelatin structure as claimed in claim 10 as carrierfor cells for the production of biological substances, comprisingintroducing such cells onto said structure, implanting said cast,three-dimensional, porous gelatin structure in the individual andallowing the cells on said structure to produce said substances.
 23. Amethod for implanting in an individual a cast, three dimensional, porousgelatin structure as claimed in claim 19 as carrier for cells for theproduction of biological substances, comprising introducing such cellsonto structure, implanting said cast, three-dimensional, porous gelatinstructure in the individual and allowing the cells on said structure toproduce said substances.
 24. A method for implanting in an individual aporous gelatin material as claimed in claim 9, comprising implantingsuch material at a site in need of treatment, and allowing thesurrounding cells to migrate to said site and colonize thereat, such asfor smoothing out wrinkles.
 25. A method for implanting in an individuala cast, three-dimensional, porous gelatin structure as claimed in claim18, comprising implanting such material at a site in need of treatment,and allowing the surrounding cells to migrate to said site and colonizethereat, such as for smoothing out wrinkles.
 26. A method for implantingin an individual a cast, three-dimensional, porous gelatin structure asclaimed in claim 10, comprising implanting such structure at a site inneed of treatment, and allowing the surrounding cells to migrate to saidsite and colonize thereat, such as for smoothing out wrinkles.
 27. Amethod for implanting in an individual a cast, three-dimensional, porousgelatin structure as claimed in claim 19, comprising implanting suchstructure at a site in need of treatment, and allowing the surroundingcells to migrate to said site and colonize thereat, such as forsmoothing out wrinkles.
 28. A method for improving in vivo healing ofdamaged tissue, comprising introducing appropriate cells onto a porousgelatin material as claimed in claim 9, and implanting said material orcast at a site of damaged tissue.
 29. A method for improving in vivohealing of damaged tissue, comprising introducing appropriate cells ontoa porous gelatin material as claimed in claim 18, and implanting saidmaterial or cast at a site of damaged tissue.
 30. A method for improvingin vivo healing of damaged tissue, comprising introducing appropriatecells onto a cast, three-dimensional, porous gelatin structure asclaimed in claim 10 and implanting said material or cast at a site ofdamaged tissue.
 31. A method for improving in vivo healing of damagedtissue, comprising introducing appropriate cells onto a cast,three-dimensional, porous gelatin structure as claimed in claim 19 andimplanting said material or cast at a site of damaged tissue.
 32. Amethod for improving in vivo healing of damaged tissue, comprisingimplanting a porous gelatin material as claimed in claim 9 at a site ofdamaged tissue and allowing the individuals own cells to proliferate onsaid material or cast.
 33. A method for improving in vivo healing ofdamaged tissue, comprising implanting a porous gelatin material asclaimed in claim 18 at a site of damaged tissue and allowing theindividuals own cells to proliferate on said material or cast.
 34. Amethod for improving in vivo healing of damaged tissue, comprisingimplanting, or a cast, three-dimensional, porous gelatin structure asclaimed in claim 10 at a site of damaged tissue and allowing theindividuals own cells to proliferate on said material or cast.
 35. Amethod for improving in vivo healing of damaged tissue, comprisingimplanting a cast, three-dimensional, porous gelatin structure asclaimed in claim 19 at a site of damaged tissue and allowing theindividuals own cells to proliferate on said material or cast.